IGF binding proteins

ABSTRACT

IGFBP-3 fusion proteins are provided that are useful, for example, in cell-based assays, as IGF antagonists, and in mapping IGF-I and IGF-II binding sites on other molecules such as wild-type IGFBP-3 and IGF agonist peptides identified by phage display. Methods for making such fusion proteins are also provided.

RELATED APPLICATIONS

This is a divisional application of non-provisional application Ser. No.10/936,059, filed on Sep. 8, 2004, now U.S. Pat. No. 7,192,738, issuedMar. 20, 2007, claiming priority under 35 USC 119(e) to provisionalapplication No. 60/508,345 filed Oct. 3, 2003, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to molecules useful in determining minimalfunctional regions of IGFBP-3 and for antagonizing an IGF-I or IGF-IIactivity.

2. Description of Background and Related Art

The insulin-like growth factors I and II (IGF-I and IGF-II,respectively) mediate multiple effects in vivo, including cellproliferation, cell differentiation, inhibition of cell death, andinsulin-like activity (reviewed in Clark and Robinson, Cytokine GrowthFactor Rev., 7: 65-80 (1996); Jones and Clemmons, Endocr. Rev., 16: 3-34(1995)). Most of these mitogenic and metabolic responses are initiatedby activation ofthe IGF-I receptor, an α₂β₂-heteroteramer closelyrelated to the insulin receptor (McInnes and Sykes, Biopoly., 43:339-366 (1998); Ullrich et al., EMBO J., 5: 2503-2512 (1986)). The IGF-Iand insulin receptors bind their specific ligands with nanomolaraffinity. IGF-I and insulin can cross-react with their respectivenon-cognate receptors, albeit at a 100-1000-fold lower affinity (Jonesand Clemmons, supra). The crystal structure describing part of theextracellular portion of the IGF-I receptor has been reported (Garrettet al., Nature, 394: 395-399 (1998)).

Unlike insulin, the activity and half-life of IGF-I are modulated by sixIGF-I binding proteins (IGFBPs 1-6), and perhaps additionally by a moredistantly related class of proteins (Jones and Clemmons, supra; Baxteret al., Endocrinology, 139: 4036 (1998)). IGFBPs can either inhibit orpotentiate IGF activity, depending on whether they are soluble orcell-membrane associated (Bach and Rechler, Diabetes Reviews, 3: 38-61(1995)). The IGFBPs bind IGF-I and IGF-II with varying affinities andspecificities (Jones and Clemmons, supra; Bach and Rechler, supra). Forexample, IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereasIGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than theybind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology, 132:1337-1344 (1993)). WO 01/75064 discloses additional human secretedIGFBP-like polypeptides that are encoded by nucleic acid sequencesisolated from cDNA libraries from adrenal gland mRNA and thymus mRNA.

Structurally, IGF-I is a single-chain, 70-amino-acid protein with highhomology to proinsulin. Unlike the other members of the insulinsuperfamily, the C region of IGF's is not proteolytically removed aftertranslation. The solution NMR structures of IGF-I (Cooke et al.,Biochemistry, 30: 5484-5491 (1991); Hua et al., J. Mol. Biol; 259:297-313 (1996)), mini-IGF-I (an engineered variant lacking the C-chain;DeWolf et al., Protein Science, 5: 2193-2202 (1996)), and IGF-II(Terasawa et al., EMBO J; 13: 5590-5597 (1994); Torres et al., J. Mol.Biol., 248: 385-401 (1995)) have been reported. It is generally acceptedthat distinct epitopes on IGF-I are used to bind receptor and bindingproteins. It has been demonstrated in animal models thatreceptor-inactive IGF mutants are able to displace endogenous IGF-I frombinding proteins and thereby generate a net IGF-I effect in viva(Loddick et al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998);Lowman et al., Biochemistry, 37: 8870-8878 (1998); U.S. Pat. Nos.6,121,416 and 6,251,865). While residues Y24, Y29, Y31, and Y60 areimplicated in receptor binding, IGF mutants thereof still bind to IGFBPs(Bayne et aL, J. Biol. Chem., 265: 15648-15652 (1990); Bayne etal., J.Biol. Chem., 264: 11004-11008 (1989); Cascieri et al., Biochemistry, 27:3229-3233 (1988); Lowman et al., sup ra).

Additionally, a variant designated (1-27,gly⁴, 38-70)-hIGF-I, whereinresidues 28-37 of the C region of human IGF-I are replaced by afour-residue glycine bridge, has been discovered that binds to IGFBP=sbut not to IGF receptors (Bar et al., Endocrinology, 127: 3243-3245(1990)).

Additionally, a variant designated (1-27,gly⁴, 38-70)-hIGF-I, whereinresidues 28-37 of the C region of human IGF-I are replaced by afour-residue glycine bridge, has been discovered that binds to IGFPB'sbut not to IGF receptors (Bar et al., Endocrinology, 127: 3243-3245(1990).

In an attempt to characterize the binding contributions of exposed aminoacid residues in the N-terminal helix, several alanine mutants of IGF-Iwere constructed (Jansson et al., Biochemistry, 36: 4108-4117 (1997)).However, the circular dichroism spectra of these mutant proteins showedstructural changes compared to wild-type IGF-I, making it difficult toclearly assign IGFBP-binding contributions to the mutated side chains. Adifferent approach was taken in a very recent study where the IGFBP-1binding epitope on IGF-I was probed by heteronuclear NMR spectroscopy(Jansson et al., J. Biol. Chem., 273: 24701-24707 (1998)). The authorsadditionally identified residues R36, R37 and R50 to be functionallyinvolved in binding to IGFBP-1.

Other IGF-I variants have been disclosed. For example, in the patentliterature, WO 96/33216 describes a truncated variant having residues1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-Isuperagonists that are derivatives of the naturally occurringsingle-chain IGF-I having an abbreviated C domain. The IGF-I analogs areof the formula: BC^(n), A wherein B is the B domain of IGF-I or afunctional analog thereof, C is the C domain of IGF-I or a functionalanalog thereof, n is the number of amino acids in the C domain and isfrom about 6 to about 12, and A is the A domain of IGF-I or a functionalanalog thereof.

Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988)discloses four mutants of IGF-I, three of which have reduced affinity tothe Type 1 IGF receptor. These mutants are: (Phe²³, Phe²⁴, Tyr²⁵) IGF-I(which is equipotent to human IGF-I in its affinity to the Types 1 and 2IGF and insulin receptors), (Leu²⁴) IGF-I and (Ser²⁴) IGF-I (which havea lower affinity than IGF-I to the human placental Type 1 IGF receptor,the placental insulin receptor, and the Type 1 IGF receptor of rat andmouse cells), and desoctapeptide (Leu²⁴) IGF-I (in which the loss ofaromaticity at position 24 is combined with the deletion of thecarboxyl-terminal D region of hIGF-I, which has lower affinity than(Leu²⁴)IGF-I for the Type 1 receptor and higher affinity for the insulinreceptor). These four mutants have normal affinities for human serumbinding proteins.

Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses threestructural analogs of IGF-I: (1-62) IGF-I, which lacks thecarboxyl-terminal 8-amino-acid D region of IGF-I; (1-27,Gly⁴, 38-70)IGF-I, in which residues 28-37 of the C region of IGF-I are replaced bya four-residue glycine bridge; and (1-27,Gly⁴, 38-62) IGF-I, with a Cregion glycine replacement and a D region deletion. Peterkofsky et al.,Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly⁴mutant of Bayne et al., supra, Vol. 264. U.S. Pat. No. 5,714,460 refersto using IGF-I or a compound that increases the active concentration ofIGF-I to treat neural damage.

Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses threeIGF-I analogs in which specific residues in the A region of IGF-I arereplaced with the corresponding residues in the A chain of insulin. Theanalogs are: (Ile⁴¹, Glu⁴⁵, Gln⁴⁶, Thr⁴⁹, Ser⁵⁰, Ile⁵¹, Ser⁵³, Tyr⁵⁵,Gln⁵⁶)IGF-I, an A chain mutant in which residue 41 is changed fromthreonine to isoleucine and residues 42-56 of the A region are replaced;(Thr⁴⁹, Ser⁵⁰, Ile⁵¹)IGF-I; and (Tyr⁵⁵, Gln⁵⁶)IGF-I.

Sliecker et al., Adv. Experimental Med. Biol., 343: 25-32 (1994))describes the binding affinity of various IGF and insulin variants toIGFBPs, IGF receptor, and insulin receptor.

IGFBPs are secreted by cells in culture and either inhibit or enhanceIGF-stimulated functions (Clemmons et aL, (1991) In Modern Concepts ofInsulin-like Growth Factors. E. M. Spencer, editor. Elsevier, New York,N.Y. 475-486). Known forms of IGFBPs include IGFBP-1, having a molecularweight of approximately 30-40 kDa in humans. See, e.g., WO89/09792,published Oct. 19, 1990, pertaining to cDNA sequences and cloningvectors for IGFBP-1 and IGFBP-2; WO89/08667, published Sep. 21, 1989,relating to an amino acid sequence of IGFBP-1; and WO89/09268, publishedOct. 5, 1989, relating to a cDNA sequence of IGFBP-1 and methods ofexpression for IGFBP-1.

IGFBP-2 has a molecular weight of approximately 33-36 kDa. See, e.g.,Binkert et al., The EMBO Journal, 8: 2497-2502 (1989), relating to anucleotide and deduced amino acid sequence for IGFBP-2.

IGFBP-3 has a non-glycosylated molecular weight of about 28 kDa. See,e.g., Baxter et al., Biochim. Biophys. Res. Com., 139:1256-1261 (1986),pertaining to a glycosylated 53-kDa subunit of IGFBP-3 that was purifiedfrom human serum; Wood et al., Mol. Endocrinol., 2:1176-1185 (1988),relating to a full-length amino acid sequence for IGFBP-3 and cellularexpression ofthe cloned IGFBP-3 CDNA in mammalian tissue culture cells;WO 90/00569, published Jan. 25, 1990, relating to isolating from humanplasma an acid-labile subunit (ALS) of IGFBP complex and the particularamino acid sequence for ALS pertaining to a subunit of IGFBP-3; andSchmid et al., Biochim. Biophys. Res Com., 179: 579-585 (1991), relatingto effects of full-length and truncated IGFBP-3 on two differentosteoblastic cell lines.

Although initially some inconsistencies in nomenclature for IGFBP-4,IGFBP-5, and IGFBP-6 existed, in 1991 participants of the 2ndInternational IGF Symposium agreed upon an accepted IGFBP-4, IGFBP-5,and IGFBP-6 nomenclature. Using accepted terminology, Mohan et al.,Proc. Natl. Acad. Sci., 86:8338-8342 (1989) relates to an N-terminalamino acid sequence for an IGFBP-4 isolated from medium conditioned byhuman osteosarcoma cells, and Shimasaki et al., Mol. Endocrinology,4:1451-1458 (1990) pertains to IGFBP cDNAs encoding IGFBP-4 from rat andhuman. WO92/03471 published Mar. 5, 1992, relates to an IGFBP-4(originally designated therein as IGFBP-5); and WO92/03470 publishedMar. 5, 1992 relates to genetic material encoding IGFBP-4 (originallydesignated therein as IGFBP-5).

WO92/12243 published Jul. 23, 1992, relates to IGFBP-5 (originallydesignated therein as IGFBP-6). Andress and Birnbaum, Biochim. Biophys.Res Com., 176: 213-218 (1991) relates to the modulation of cellularaction of a mixture of affinity-purified IGFBPs fromU-2-cell-conditioned media on IGFs. WO92/03469 published Mar. 5, 1992,relates to genetic material encoding IGFBP-6 (originally designatedtherein as IGFBP-4); and WO92/03152 published Mar. 5, 1992, relates toan IGFBP-6 (originally designated therein as IGFBP-4). See also USappln. No. 2003/0082744 as well as U.S. Pat Nos. 6,025,465 and5,212,074, which disclose IGFBP-6 and its fragments.

Zapf et al., J. Biol. Chem., 265:14892-14898 (1990) pertains to fourIGFBPs (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, and IGFBP4)isolated from adult human serum by insulin-like growth factor (IGF)affinity chromatography and high-performance liquid chromatography.Shimasaki et al., 2nd International IGF Symposium Abstract (January1991) discusses amino-terminal amino acids for IGFBP-4, IGFBP-5, andIGFBP-6.

When administered alone, i.e., without any IGF, the IGFBPs may also betherapeutically useful for blocking the adverse effects of IGFs, such asthose that occur when IGFs are produced in excess, e.g. free IGFssecreted by certain cancer cells such as hormone-producing cancer cellssuch as breast or kidney cancer cells. More recently, it wasdemonstrated that U-2 human osteosarcoma cells secrete IGFBP-5 andIGFBP-6 (Andress and Birnbaum, supra; Shimasaki et al., J. Biol. Chem.,266: 10646-10653 (1991); Shimasaki et al., Mol. Endocrinol., 5: 938-948(1991)). Although affinity-purified IGF-binding proteins derived fromU-2-conditioned medium clearly enhanced IGF-I stimulated mitogenesis(Andress and Birnbaum, supra), it was unclear from those studies whichprotein was responsible for this effect. Mohan et al. demonstrated thatIGFBP-4, purified from TE-89 human osteosarcoma cells, inhibitsIGF-stimulated osteoblast mitogenesis (Mohan et al., Proc. Natl. Acad.Sci. (U.S.A.), 86: 8338-8342 (1989); see also LaTour et al., Mol.Endocrinol., 4: 1806-1814 (1990)). WO 03/068160 discloses use of IGFBP-3for inhibiting tumor growth. WO 03/006029 discloses a method of inducingapoptosis in a cancer cell comprising increasing the expression ofIGFBP-5 by the cell to an apoptosis-inducing amount. A method of killingcancer cells, a method of sensitizing cancer cells to agents that induceapoptosis, and a method of treating cancer in a patient are alsodescribed. U.S. Pat. No. 6,410,335 discloses a method of predicting riskfor prostate cancer, and US application 2001/0018190 published Aug. 30,2001 discloses a method of treating prostate cancer with, inter alia,IGFBPs, including IGFBP-3. U.S. Pat. No. 5,840,673 and EP 871,475disclose a method of inhibiting growth of p53-related tumors byadministering IGFBP-3 or a modulator of IGFBP-3 that upregulates IGFBP-3expression or activity. WO 00/35473 discloses use of IGFBP-6 to treatinflammatory diseases including tumor angiogenesis. WO 94/22466discloses use of any IGFBP-3 to treat cancer, including prostate cancer.WO 92/14834 discloses two IGFBPs isolated from rat serum, one identifiedas IGFBP-5 useful in treating cancer.

Exploitation of the interaction between IGF and IGFBP in screening,preventing, or treating disease has been limited, however, because of alack of specific antagonists. The application of an IGF-1/IGF-2antagonist as a potential therapeutic adjunct in the treatment of canceris described by Pietrzkowski et al., Cancer Res., 52: 6447-6451 (1992).In that report, a peptide corresponding to the D-region of IGF-1 wassynthesized for use as an IGF-1/2 antagonist. This peptide exhibitedquestionable inhibitory activity against IGF-1. The basis for theobserved inhibition is unclear, as the D-region does not play asignificant role in IGF-1 receptor (IGF-1R) binding but rather, in IGF-1binding to the insulin receptor (Cooke et al., Biochem., 30: 5484-5491(1991); Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988); Yee etal., Cell Growth and Different., 5: 73-77 (1994)). IGF antagonists whosemechanism of action is via blockade of interactions at the IGF-1receptor interface may also significantly alter insulin action at theinsulin receptor, a disadvantage of such antagonists.

Certain IGF-1 antagonists have also been described by WO 00/23469, whichdiscloses the portions of IGFBP and IGF peptides that account for IGF-1and IGFBP binding, i.e., an isolated IGF binding domain of an IGFBP ormodification thereof that binds IGF with at least about the same bindingaffinity as the full-length IGFBP. The patent publication also disclosesan IGF antagonist that reduces binding of IGF to an IGF receptor, and/orbinds to a binding domain of IGFBP.

Additionally, EP 639981 discloses pharmaceutical compositions comprisingshort peptides that function as IGF-1receptor antagonists. The peptidesused in the pharmaceutical compositions consist of less than 25 aminoacids, comprise at least a portion of the C or D region from IGF-1, andinhibit IGF-1-induced autophosphorylation of IGF-1 receptors. Methods ofinhibiting cell proliferation and of treating individuals suspected ofsuffering from or susceptible to diseases associated with undesirablecell proliferation such as cancer, restenosis, and asthma are disclosed.

Generation of specific IGF-1 antagonists has been restricted, at leastin part, because of difficulties in studying the structure of IGF andIGFBP. Due to the inability to obtain crystals of IGF-1 suitable fordiffraction studies, for example, an extrapolation of IGF-1 structurebased on the crystal structure of porcine insulin was the most importantstructural road map for IGF-1 available (Blundell et al., Proc. Natl.Acad. Sci. USA, 75:180-184 (1978)). See also Blundell et al., Fed Proc.,42: 2592 (1983), which discloses tertiary structures, receptor binding,and antigenicity of IGFs. Based on studies of chemically modified andmutated IGF-1, a number of common residues between IGF-1 and insulinhave been identified as being part of the IGF-1R-insulin receptorcontact site, in particular the aromatic residues at positions 23-25.Using NMR and restrained molecular dynamics, the solution structure ofIGF-1 was recently reported (Cooke et al., supra). The resultingminimized structure was shown to better fit the experimental findings onmodified IGF-1, as well as the extrapolations made from thestructure-activity studies of insulin. Further, De Wolf et al., ProteinSci., 5: 2193 (1996) discloses the solution structure of a mini-IGF-1.Sato et al., Int. J. Pept., 41: 433 (1993) discloses thethree-dimensional structure of IGF-1 determined by 1 H-NMR and distancegeometry. Torres et al., J Mol. Biol., 248: 385 (1995) discloses thesolution structure of human IGF-2 and its relationship to receptor andbinding protein interactions. Laajoki et al., J. Biol. Chem., 275: 10009(2000) discloses the solution structure and backbone dynamics oflong-(Arg(3)) IGF-1.

Peptide sequences capable of binding to insulin and/or insulin-likegrowth factor receptors with either agonist or antagonist activity andidentified from various peptide libraries are described in WO 01/72771published Oct. 4, 2001.

US Application No. 2003/0092631 published May 15, 2003 disclosespeptides that antagonize the interaction of IGF-1 with its bindingproteins, insulin receptor, and IGF receptor. These IGF antagonistpeptides are useful in treating disorders involving IGF-1 as a causativeagent, such as, for example, various cancers.

Regarding the structural information on the classical IGFBPs, they havea molecular mass ranging from 22 to 31 kDa and contain a total of 16-20cysteines in their conserved amino- and carboxy-terminal domains (Bachand Rechler, supra; Clemmons, Cytokine Growth Factor Rev., 8: 45-62(1997); Martin and Baxter, Curr. Op. Endocrinol. Diab., 16-21 (1994)).The central domain connecting both cysteine-rich regions is only weaklyconserved and contains the cleavage sites for IGFBP-specific proteases(Chemausek et al., J. Biol. Chem., 270: 11377-11382 (1995); Clemmons,supra; Conover, Prog. Growth Factor Res., 6: 301-309 (1995)). Furtherregulation of the IGFBPs may be achieved byphosphorylation andglycosylation (Bach and Rechlersupra; Clemmons, supra). There is nohigh-resolution structure available for any intact member of the IGFBPfamily. U.S. Pat. No. 6,500,635 discloses IGFBP-5 and its variants. U.S.Pat. Nos. 6,391,588 and 6,489,294 disclose truncated C-terminal IGFBP-5fragments with reduced affinity for IGF-I as compared to full-lengthIGFBP-5. U.S. Pat. No. 6,369,029 discloses stimulating osteogenesis withC-terminal-truncated IGFBP-5. These compounds may be used forstimulating bone cell growth, for treating a bone disorder, or forstimulating mitogenic activity.

The NMR structures of two N-terminal fragments from IGFBP-5 that retainIGF-binding activity have recently been reported, showing that residues40-92 of IGFBP-5 comprise the IGF binding site in the N-terminal domainof that protein (Kalus et al., EMBO J., 17: 6558-6572 (1998)). Otherstudies have found that N-terminal fragments (residues 1-88 and 1-97) ofIGFBP-3 are also able to bind IGFs (Galanis et al., Journal ofEndocrinology, 169 (1): 123-133 (2001); Vorwerk et al., Endocrinolgy,143 (5): 1677-1685 (2002

In particular, Galanis et al. have synthesized both the amino-terminal(residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165)domains of human IGFBP-3 in bacteria, as fusion proteins with acarboxyl-terminal FLAG peptide. Although only C-165 showed binding toIGF-I and -II by solution-binding assays, both N-88 and C-165demonstrated binding to IGF-I and -II by biosensor analysis albeit withreduced affinities compared with full-length IGFBP-3. Only thecarboxyl-terminal fragment (C-165) was able to form hetero-trimericcomplexes with IGF-I and the acid-labile subunit (ALS).

Vorwerk et al. measured the binding of IGF-I and IGF-II to recombinanthuman N-terminal (residues 1-97; N-97) and C-terminal (residues 98-264;C-98) IGFBP-3 fragments and compared it with IGF binding to intactIGFBP-3 using biosensor analysis. Experiments were carried out eitherwith binding protein or fragment immobilized or with IGF immobilized.These experiments showed that IGF-I and IGF-II bind to IGFBP-3 withaffinities of 4-5×10⁻⁹ M and similar binding kinetics. The affinities ofboth N-97 and C-98 for IGF proteins were approximately three orders ofmagnitude less than that of full-length IGFBP-3.

US Application No. 2003/0161829A 1 published Aug. 28, 2003 and WO03/025121 disclose fragments of IGFBP-3 that do not bind IGF-I to treatconditions characterized by immune stimulation rather than deficiency.The peptides target the CD74-homology domain sequence at the C-terminusof IGFBP-3 and activities localized to that region, having uniqueantigenicity. Peptides made to sequences in this region have previouslybeen shown to interfere with the binding of IGFBP-3 to a number of itsknown ligands, including RXR-alpha, transferrin, ALS, plasminogen,fibrinogen and pre-kallikrein (Liu et al., J. Biol. Chem., 275:33607-33613 (2000); Weinzimer et al., J. Clin. Endocrinol. Metab., 86:1806-13 (2001); Campbell et al., Am. J. Physiol., 275: E321-E231 (1998); Campell et al., J. Biol. Chem., 274: 30215-30221 (1999); Firth, etal., J. Biol. Chem., 273: 2631-2638, (1998

The IGFBP-3-derived metal-binding domain peptides differ from previouslydisclosed IGFBP-3-derived molecules including in their inability to bindIGF-I their unique antigenicity, and the absence of the IGFBP-3 putativedeath receptor (P4.33) interaction domain of IGFBP-3 (so-called“mid-region”; amino acids 88-148). The P4.33 putative death receptor isdescribed in WO 01/87238 (Genbank Accession Number BC031217;gi:21411477). For example, WO 02/34916 teaches the use of point mutantsof IGFBP-3 in which the binding to IGF-I is impaired. However, thedescribed molecules contain the mid-region of IGFBP-3 and would beexpected to exert biological effects by interacting with the P4.33putative receptor. WO 01/87238 teaches the use of P4.33 modulators fortreating disease. The metal-binding peptides do not include the P4.33putative interaction domain (mid-region of IGFBP-3).

U.S. Pat. No. 6,417,330, WO 99/63086, and US application no.2002/0072589 disclose IGFBP-3 variants modified to be resistant tohydrolysis. Also disclosed are variant IGFBP-3s where the nuclearlocalization signal (NLS) in native IGFBP-3 is altered. Additionally,amino-terminally extended IGFBP-3s are disclosed that include a varietyof N-terminal extensions, including peptide and nucleotide bindingdomains, specific binding members such as ligand-binding domains fromreceptors or antigen-binding domains from immunoglobins, and peptide andprotein hormones and growth factors. N-terminally extended IGFBP-3s maycomprise hydrolysis-resistant or NLS-variant IGFBP-3s.

Some recent publications have described the use of IGFBP-3 peptides totreat cells in culture. The only peptides found to be active on breastcancer cells are derived from the mid-region of IGFBP-3 (McCaig et al.,Br. J. Cancer, 86: 1963-1969 (2002); Perks et al., Biochim. Biophys.Res. Comm., 294: 988-994 (2002)).

US Application No. 2003/0059430 published Mar. 27, 2003 discloses thatthe IGF-binding protein-derived peptides described above, includingshort peptides containing just 12-22 amino acids from the C-terminaldomain of IGFBP-3, can mimic the full molecule's co-apoptotic,cell-penetrating, and metal-binding properties.

WO 03/052079 discloses mutants of IGFBP-3 that can inhibit DNAsynthesis, can induce apoptosis, bind to neither human IGF-I nor humanIGF-II, and comprise a mutation at Y57.

WO 02/098914 discloses a crystal suitable for X-ray diffraction,comprising a complex of IGF-I or -II and a polypeptide consisting of theamino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, theamino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP4, the aminoacids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or afragment thereof consisting at least of the 9th to 12th cysteine ofIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to10th cysteine of IGFBP-6; methods for the determination of the atomiccoordinates of such a crystal; IGFBP mutants with enhanced bindingaffinity for IGF-I and/or IGF-II, and methods to identify and optimizesmall molecules that displace IGFs from their binding proteins.

WO 02/34916 discloses mutant IGFBP-3 polypeptides and fragments there ofthat have either no binding, or diminished binding to IGFs, yet retaintheir ability to bind to the human IGFBP-3 receptor (“P4.33”). Thefragments are N-deletion fragments with 87 to 264 amino acids. Thefragment 1-87 binds poorly to IGF-I, and the other fragments (1-46,1-75, and 1-80) do not bind at all.

WO 00/23469 discloses IGFBP fragments that account for IGF-IGFBPbinding. It provides an isolated IGF binding domain of an IGFBP ormodifications thereof, which binds IGF with at least about the samebinding affinity as the full-length IGFBP. It also provides an IGFantagonist that reduces binding of IGF to an IGF receptor. It especiallyrelates to IGFBP-2 fragments, but also provides the isolated IGF bindingdomains of IGFBP-1, IGFBP-3, IGFBP4, IGFBP-5, and IGFBP-6. As with theIGF binding domain of IGFBP-2, the amino acid sequences comprising theIGF binding domain of the other IGFBPs could include modified forms solong as the binding affinity of the binding domain is about the same asthat of the comparable native fill-length IGFBP.

WO 99/32620 discloses IGFBP fragments and utilization thereof, i.e.,peptides that are characterized in that the amino acid sequence partsthereof correspond to the amino acid sequence of IGFBP. The inventionalso relates to cyclic, glycosylated, phosphorylated, acetylated,amidated and/or sulfatized derivatives. These include C-terminal domainsof IGFBP-3.

The use of gene fusions, though not essential, can facilitate theexpression of heterologous peptides in E. coli as well as the subsequentpurification of those gene products (Harris, in Genetic Engineering,Williamson, R., Ed. (Academic Press, London, Vol. 4, 1983), p.127;Ljungquist et al., Eur.J. Biochem., 186: 557-561 (1989); and Ljungquistet al., Eur. J. Biochem., 186: 563-569 (1989)). Protein A fusions areoften used because the binding of protein A, or more specifically the Zdomain of protein A, to IgG provides an “affinity handle” for thepurification of the fused protein. It has also been shown that manyheterologous proteins are degraded when expressed directly in E. coli,but are stable when expressed as fusion proteins (Marston, Biochem J.,240: 1 (1986)).

Fusion proteins can be cleaved using chemicals, such as cyanogenbromide, which cleaves at a methionine, or hydroxylamine, which cleavesbetween an Asn and Gly residue. Using standard recombinant DNAmethodology, the nucleotide base pairs encoding these amino acids may beinserted just prior to the 5′ end of the gene encoding the desiredpeptide.

Alternatively, one can employ proteolytic cleavage of fusion proteins(Carter, in Protein Purification: From Molecular Mechanisms toLarge-Scale Processes, Ladisch et al., eds. (American Chemical SocietySymposium Series No. 427, 1990), Ch 13, pages 181-193)).

There is a continuing need in the art for a molecule that can be used toelucidate binding epitopes on IGFBP-3 and other ligands, acts as an IGFantagonist to control the levels of circulating IGF as well as receptorresponse, for therapeutic or diagnostic uses, and can be used for othertherapeutic, diagnostic, or assay purposes.

SUMMARY OF THE INVENTION

Accordingly, the invention is as claimed. In one embodiment, thisinvention provides a fusion protein comprising an IGFBP-3 fragment(i.e., a truncated IGFBP-3) consisting of residues 47 to 99 ofnative-sequence human IGFBP-3 (SEQ ID NO:1below) linked to thesynthesized Z domain of protein A from Staphylococcus aureus, which hasthe sequence:

VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKD (SEQ ID NO:10)DPSQSANLLAEAKKLNDAQAPK.

In one such embodiment, this fusion protein is displayed on phage. Inanother embodiment, the fragment is linked to the Z domain by means of acleavable linker peptide. Such cleavable linker peptide preferablycomprises one of the sequences: DLVD (SEQ ID NO:2), DEMD (SEQ ID NO:3),DAVD (SEQ ID NO:4), EFGGDDDK (SEQ ID NO;5), EFGGLVPRGS (SEQ ID NO:6),EFGGDLVD (SEQ ID NO:7), EFGGDEMD (SEQ ID NO:8), or EFGGDAVD (SEQ IDNO:9). In another embodiment, an ASA sequence is at the N-terminus ofthe Z domain. In yet another embodiment, the fragment is affinitymatured.

Also provided herein is a composition comprising the fusion proteindescribed above in a carrier, preferably a pharmaceutically acceptablecarrier. Preferably, this composition is sterile.

Further provided is a nucleic acid molecule encoding the fusion protein,a vector comprising the nucleic acid, a host cell comprising the nucleicacid, and a method of producing an IGFBP-3 fusion protein comprisingculturing the host cells under suitable conditions to express the fusionprotein and recovering the fusion protein from the host cell culture.Preferred is that the host cells are prokaryotic, more preferablybacterial cells such as E. coli.

These fusion proteins can be used in many indications, including assaysfor determining if ligands have an IGFBP-3 binding site. The fusionproteins herein containing an IGF-I or IGF-II binding site are furtheruseful for clarification and mapping of the IGF binding sites andbinding sites of IGF-I agonist ligands other than IGF-I (e.g., peptidesisolated by phage panning experiments such as bp15 described in Lowmanet al., supra) on native-sequence human IGFBP-3 in the absence of anystructural data.

In yet another embodiment, the invention provides a method fordetermining a biological activity of native-sequence human IGFBP-3, ofnative-sequence human IGF-I, or of native-sequence human IGF-II, or ofan agonist of said IGF-I or said IGF-II in a cell-based assay comprisingcontacting cells with a fusion protein comprising a peptide linked to anIGFBP-3 fragment that binds to IGF-I or IGF-II, rather than withnative-sequence human IGFBP-3, and determining if a biological activityattributable to native-sequence human IGFBP-3, native-sequence humanIGF-I, or native-sequence human IGF-II, or an agonist of said IGF-I orsaid IGF-II is observed.

In one embodiment, the biological activity is apoptosis ofnative-sequence human IGFBP-3 that is independent of IGF-I.

In another embodiment, the assay is an IGF-dependent KIRAphosphorylation assay. This assay is a direct activity assay for thehuman Type 1 receptor. When a receptor in the tyrosine kinase family,such as the Type 1 IGF receptor, is activated, it is phosphorylated ontyrosine residues. In this assay cells containing the Type 1 IGFreceptor are activated in vitro, then disrupted, and antibodies againstthe receptor are used to precipitate the IGF receptor. Next, ananti-phosphotyrosine antibody is used to assay the amount of Type 1 IGFreceptor that is phosphorylated. If a fixed number of cells are used,then the amount of receptor that is phosphorylated is a direct measureof the activity of a molecule on the Type 1 IGF receptor. In this KIRAassay, cells such as a breast cancer cell line are treated with IGF-I orIGF-II plus the fusion protein and a biological activity of the fusionprotein is determined by the amount of receptor that is phosphorylated.

In yet another embodiment, the biological activity is inhibition ofbinding ofradiolabeled IGF-I or IGF-II to the cells.

In a further aspect, the invention provides a method for determiningenhancement of apoptosis comprising pre-treating breast cancer cellswith an IGFBP-3 fragment or fusion protein thereof that binds IGF-I orIGF-II for at least about 24 hours prior to treating the cells with anapoptotic factor, such as, for example, paclitaxel or doxorubicin, andnative-sequence human IGFBP-3 or said IGFBP-3 fragment or fusionprotein, and determining if the pre-treatment or treatment enhances theapoptosis induced by the treatment with the apoptotic factor, or if theamounts of pre-treatment or treatment are effective for that purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the miniBP-3 fusion protein. This is thefusion of a Z domain (SEQ ID NO:10) to miniBP-3 (residues 47-99 ofnative-sequence human IGFBP-3 (SEQ ID NO:1 )) using a cleavable linkerhaving SEQ ID NO:7 (including the caspase-3 cleavage site (SEQ ID NO:2),which is underlined).

FIG. 2 shows a SDS-PAGE analysis, using Coomassie blue staining, ofvarious chromatographic fractions of the miniBP-3 fusion protein and thecleaved protein miniBP-3.

FIG. 3 shows a biosensor analysis of native-sequence human IGF-I bindingto immobilized native-sequence human IGFBP-3.

FIG. 4 shows a biosensor analysis of native-sequence human IGF-IIbinding to immobilized native-sequence human IGFBP-3.

FIG. 5 shows a biosensor analysis of native-sequence human IGF-I bindingto immobilized miniBP-3 fusion protein.

FIG. 6 shows a biosensor analysis of native-sequence human IGF-IIbinding to immobilized miniBP-3 fusion protein.

FIG. 7 shows the results of peptide bp15 (SEEVCWPVAEWYLCN) (SEQ ID NO:11) competition experiments performed on the BIAcore™ analyzer. A total of15 nM to 100:M bp15 peptide and 20 nM native-sequence human IGFBP-3 wereincubated for 1 hour at room temperature before injection overimmobilized biotinylated native-sequence human IGF-I and -II.

FIG. 8 shows the results of peptide bp15 (SEQ ID NO:11) competitionexperiments performed on the BIAcore™ analyzer. A total of 1.17:M to300:M bp15 peptide and 1:M miniBP-3 fusion protein was incubated 1 hourat room temperature before injection over immobilized biotinylatednative-sequence human IGF-I.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Definitions

As used herein, “IGF” refers to native insulin-like growth factor-I andnative insulin-like growth factor-II as well as natural variants thereofsuch as brain IGF, otherwise known as des(1-3)IGF-I.

As used herein, “IGF-I” refers to insulin-like growth factor-I from anyspecies, including bovine, ovine, porcine, equine, and human, preferablyhuman, and from any source, whether natural, synthetic, or recombinant.This may be prepared, e.g., by the process described in EP 230,869published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; or EP288,451 published Oct. 26, 1988. “Native-sequence human IGF-I” or“wild-type IGF-I” is wild-type human IGF-I.

As used herein, “IGF-II” refers to insulin-like growth factor-II fromany species, including bovine, ovine, porcine, equine, and human,preferably human, and from any source, whether natural, synthetic, orrecombinant. It may be prepared by the method described in, e.g., EP128,733. “Native-sequence human IGF-II” or “wild-type IGF-II” iswild-type human IGF-II.

An AIGFBP@ or an “IGF binding protein” refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-I orIGF-II, whether or not it is circulatory (i.e., in serum or tissue).Such binding proteins do not include receptors. This definition includesIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7),and prostacyclin-stimulating factor (PSF) or endothelial cell-specificmolecule (ESM-1), as well as other proteins with high homology toIGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc.Natl. Acad. Sci. USA, 92:4472-4476 (1995) and Oh et al., J. Biol. Chem.,271: 30322-30325 (1996). PSF is described in Yamauchi et al.,Biochemical Journal, 303: 591-598(1994). ESM-1 is described in Lassalleet al., J. Biol. Chem., 271: 20458-20464 (1996). For other identifiedIGFBPs, see, e.g., EP 375,438 published 27 Jun. 1990; EP 369,943published 23 May 1990; WO 89/09268 published 5 Oct. 1989; Wood et al.,Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBOJ., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411(1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 published7 Dec. 1988; Baxter et al., BBRC, 147:408-415 (1987); Leung et al.,Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261:8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235(1988); WO 89/08667 published 21 Sep. 1989; WO 89/09792 published 19Oct. 1989; Binkert et al., EMBO J., 8: 2497-2502 (1989); EP 369,943B1;U.S. Pat. No. 5,973,115; EP 1,295,939; and U.S. Pat. No. 6,004,775 andEP 546053.

“IGFBP-3” or “insulin-like growth factor binding protein-3” refers toIGFBP-3 or BP53 from any species including bovine, ovine, porcine,equine, and human, preferably human, and from any source, whethernatural, synthetic, or recombinant, as described in U.S. Pat. Nos.5,258,287 and 5,328,891. “Native-sequence human IGFBP-3,” “wild-typeIGFBP-3,” and “full-length IGFBP-3” is wild-type human IGFBP-3 or BP-53as described in U.S. Pat. Nos. 5,258,287 and 5,328,891 with a glycine atposition 5, i.e., SEQ ID NO:1, or an alanine at position 5, i.e., SEQ IDNO:12.

SEQ ID NO:1 is the sequence:GASSGGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVSRLRAYLLPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTT KGKEDVHCYSMQSK. SEQID NO:12 is the sequence:GASSAGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVSRLRAYLLPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTT KGKEDVHCYSMQSK

“IGFBP-3 fragment” refers to native-sequence human IGFBP-3 (SEQ ID NO:1or 12) lacking at least one amino acid. Preferably, the fragment is anN-terminal fragment such as, for example, the peptide having (orcorresponding to) residues 1 to 46, residues 1 to 88, residues 1 to 89,residues 1 to 90, residues 1 to 91, residues 1 to 92, residues 1 to 93,residues 1 to 94, residues 1 to 95, residues 1 to 96, residues 1 to 97,residues 1 to 98, residues 1 to 99, residues 1 to 185, or residues 47 to99 of native-sequence human IGFBP-3 (SEQ ID NO:1 or 12), or a C-terminalfragment such as, for example, the peptide having residues 98 to 264,residues 100 to 264, residues 165 to 264, or residues 185 to 264 ofnative-sequence human IGFBP-3 (SEQ ID NO:1). More preferably, theIGFBP-3 fragment is selected from the group consisting of a peptide withresidues 1 to 46, residues 1 to 88, residues 1 to 97, residues 1 to 99,residues 47 to 99, residues 1 to 185, residues 98 to 264, residues 100to 264, residues 165 to 264, and residues 185 to 264 of native-sequencehuman IGFBP-3 (SEQ ID NO:1 or 12), and most preferably a peptide withresidues 47 to 99 of native-sequence human IGFBP-3 (SEQ ID NO:1).

A “fusion protein” is a protein having two separate peptides linkedtogether, either directly or through a linker (i.e., “linker peptide” or“linking peptide”), preferably through such a linker. For example, onefusion protein is a protein comprising the Z domain of Protein A fusedto an IGFBP-3 fragment, which Z domain and fragment may be directlylinked if no cleavage is desired, or may be linked via a cleavage sitesuch as a caspase-3 proteolytic site if cleavage is desired.

As used herein, “Z domain of protein A” refers to an IgG-binding domainof Staphylococcal protein A as described, for example, in EP 230,869 B 1as the synthetic Z-region in FIG. 3, and in Samuelsson et al.,Bio/Technology, 9: 363 (1991), and Nilsson et al., Protein Eng., 1(2):107-113 (1987).

“Peptides” are molecules having at least two amino acids and includepolypeptides having at least about 50 amino acids. The definitionincludes peptide fragments, derivatives, their salts, or opticalisomers, as well as linkers. Preferably, the peptides herein have fromabout 35 to 200 amino acids, more preferably, about 40 to 170 aminoacids.

An “apoptotic factor” as used herein is a molecule that inducesapoptosis or cell death. Such molecules include chemotherapeutic agents,anti-hormonal agents, cytotoxic agents and other agents some of whichare defined below that induce cell death. Preferably, such factor is achemotherapeutic agent, and more preferably doxorubicin or paclitaxel,most preferably paclitaxel.

An “affinity-matured” IGFBP-3 peptide fragment or fusion protein is onehaving one or more alterations within the peptide fragment or fusionprotein that result in an improvement in the affinity of the peptidefragment or fusion protein for IGF-I or IGF-II, compared to acorresponding parent peptide fragment or fusion protein that does notpossess those alteration(s). Preferred affinity-matured IGFBP-3 peptidefragment or fusion protein will have nanomolar or even picomolaraffinities for the target IGF. Affinity-matured peptide fragments andfusion proteins are produced by procedures known in the art, includingphage display (Lowman and Wells, J. Mol. Biol., 234 (3): 564-578(1993);Lowmnan et al., Biochemistry, 30 (45): 10832-10838(1991)); rationalmutagenesi (Lowman et al., J. Biol. Chem., 261 (17): 10982-10988 (1991);Hawkins et al., J. Mol. Biol., 234 (4): 958-964 (1993)); randommutagenesis (Fiedler et al., Protein Eng., 15 (11): 931-941 (2002)); andDNA shuffling and phage display (Huls et al., Cancer Immunol.Immunother., 50 (3): 163-171 (2001); van den Beucken et al., J. Mol.Biol., 310 (3): 591-601 (2001)).

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic, and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, sheep,pigs, cows, etc. The preferred mammal herein is a human. The term“non-adult” refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented. Consecutive treatment or administrationrefers to treatment on at least a daily basis without interruption intreatment by one or more days. Intermittent treatment or administration,or treatment or administration in an intermittent fashion, refers totreatment that is not consecutive, but rather cyclic in nature. Thetreatment regime herein can be either consecutive or intermittent.

As used herein, “IGF antagonist” refers to a peptide that blocks orinhibits one or more biological activities of IGF-I or IGF-II such asits anabolic effects.

As used herein, “active” or “biologically active” IGF in the context ofchanging serum and tissue levels of endogenous IGF refers to IGF thatbinds to its receptor or otherwise causes a biological activity tooccur, such as an anabolic effect.

The term “effective amount” refers to an amount of a peptide effectiveto treat a disease or disorder in a mammal. In the case of cancer, theeffective amount of the peptide may reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder. To the extent the peptide mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the time to disease progression (TTP) and/ordetermining the response rates (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD).Preferably, the cancer comprises a tumor that expresses an IGF receptor,more preferably breast cancer, lung cancer, colorectal cancer, orprostate cancer, and most preferably breast or prostate cancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At211,I131, I125, Y90, Re 186, Re188, Sm153, Bi212, P32 and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such assmall-molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gammalI and calicheamicin omegal I (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11topoisomerase inhibitor RFS2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON•toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those thatinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth-inhibitory agent” when used herein refers to a compound orcomposition that inhibits growth of a cell in vitro and/or in vivo.Thus, the growth-inhibitory agent may be one that significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce GI arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL® paclitaxel, and topo II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest GI also spill over into S-phase arrest, for example,DNA alkylating agents such as tanoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antieioplastic drugs” by Murakaini et al. (W B Saunders:Philadelphia, 1995), especially p. 13.

Examples of “growth-inhibitory” anti-HER2 antibodies are those that bindto HER2 and inhibit the growth of cancer cells overexpressing HER2.Preferred growth-inhibitory anti-HER2 antibodies inhibit growth of SKBR3breast tumor cells in cell culture by greater than 20%, and preferablygreater than 50% (e.g., from about 50% to about 100%) at an antibodyconcentration of about 0.5 to 30 μg/ml, where the growth inhibition isdetermined six days after exposure of the SKBR3 cells to the antibody(see U.S. Pat. No. 5,677,171 issued Oct. 14, 1997).

An antibody that “induces cell death” is one that causes a viable cellto become nonviable. The cell is generally one that expresses theantigen to which the antibody binds, especially where the celloverexpresses the antigen. Preferably, the cell is a cancer cell, e.g.,a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be aSKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell deathin vitro may be determined in the absence of complement and immuneeffector cells to distinguish cell death induced by antibody dependentcell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity(CDC). Thus, the assay for cell death may be performed usingheat-inactivated serum (i.e., in the absence of complement) and in theabsence of immune effector cells. To determine whether the antibody isable to induce cell death, loss of membrane integrity as evaluated byuptake of propidium iodide (PI), trypan blue (see Moore et al.Cytotechnology, 17:1-11 (1995)) or 7AAD can be assessed relative tountreated cells.

An antibody that “induces apoptosis” is one that induces programmed celldeath as determined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone that expresses the antigen to which the antibody binds and may beone that overexpresses the antigen. The cell may be a tumor cell, e.g.,a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be aSKBR3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Variousmethods are available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody that induces apoptosis is one that results inabout 2- to 50-fold, preferably about 5- to 50-fold, and most preferablyabout 10- to 50-fold, induction of annexin binding relative to untreatedcell in an annexin-binding assay using cells expressing the antigen towhich the antibody binds.

Examples of antibodies that induce apoptosis include the anti-HER2monoclonal antibodies 7F3 (ATCC HB-12216), and 7C2 (ATCC HB 12215),including humanized and/or affinity-matured variants thereof; theanti-DR5 antibodies 3F11.39.7 (ATCC HB-12456); 3H3.14.5 (ATCC HB-12534);3D5. 1.10 (ATCC HB-12536); and 3H3. 14.5 (ATCC HB-12534), includinghumanized and/or affinity-matured variants thereof; the human anti-DR5receptor antibodies 16E2 and 20E6, including affinity-matured variantsthereof (WO98/51793, expressly incorporated herein by reference); andthe anti-DR4 antibodies 4E7.24.3 (ATCC HB-12454); 4H6.17.8 (ATCCHB-12455); 1H5.25.9 (ATCC HB-12695); 4G7.18.8 (ATCC PTA-99); and 5GI1.17.1 (ATCC HB-12694), including humanized and/or affinity-maturedvariants thereof.

In order to screen for antibodies that bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratorv Manual, eds. Harlow and Lane(New York: Cold Spring Harbor Laboratory, 1988) can be performed.

B. Modes for Carrying Out the Invention

The invention herein relates, in one aspect, to a fusion proteincomprising an IGFBP-3 peptide fragment containing only residues 47-99 ofnative-sequence human IGFBP-3 (SEQ ID NO: 1), referred to herein asminiBP-3, linked to the Z domain of Protein A, whether linked directlyor through a linker, which can be cleaved by an enzyme so as to releasethe fragment. Such linker or linking peptide may be, for example, acleavable linker, e.g., a caspase-3 cleavable linker containing theproteolytic site DLVD (SEQ ID NO:2), DEMD (SEQ ID NO:3) or DAVD (SEQ IDNO:4). Examples of such linking peptides are EFGGDLVD (SEQ ID NO:7),EFGGDEMD (SEQ ID NO:8), or EFGGDAVD (SEQ ID NO:9). Other examples oflinking peptides are the enterokinase-cleavable linker EFGGDDDK (SEQ IDNO:5) and a thrombin-cleavable linker EFGGLVPRGS (SEQ ID NO:6).

1. Preparation

The fusion peptides of this invention can be made by chemical synthesisor by employing recombinant technology. These methods are known in theart. Chemical synthesis, especially solid phase synthesis, is preferredfor short (e.g., less than 50 residues) peptides or those containingunnatural or unusual amino acids such as D-Tyr, Ornithine, amino adipicacid, and the like. Recombinant procedures are preferred for longerpolypeptides. When recombinant procedures are selected, a synthetic genemay be constructed de novo or a natural gene may be mutated by, forexample, cassette mutagenesis. Set forth below are exemplary generalrecombinant procedures.

a. Recombinant Preparation

The fusion peptide may be produced using recombinant DNA techniques.These techniques contemplate, in simplified form, taking the gene,either natural or synthetic, encoding the peptide; inserting it into anappropriate vector; inserting the vector into an appropriate host cell;culturing the host cell to cause expression of the gene; and recoveringor isolating the peptide produced thereby. Preferably, the recoveredpeptide is then purified to a suitable degree.

Somewhat more particularly, the DNA sequence encoding an IGFBP-3 fusionprotein is cloned and manipulated so that it may be expressed in aconvenient host. DNA encoding parent polypeptides can be obtained from agenomic library, from cDNA derived from mRNA from cells expressing thepeptide, or by synthetically constructing the DNA sequence (Sambrook etal., Molecular Cloning: A Laboratory Manual (2d ed.), Cold Spring HarborLaboratory, N.Y., 1989).

The parent DNA is then inserted into an appropriate plasmid or vectorthat is used to transform a host cell. In general, plasmid vectorscontaining replication and control sequences that are derived fromspecies compatible with the host cell are used in connection with thosehosts. The vector ordinarily carries a replication site, as well assequences that encode proteins or peptides that are capable of providingphenotypic selection in transformed cells.

For example, E. coli may be transformed using pBR322, a plasmid derivedfrom an E. coli species (Mandel et al., J. Mol. Biol., 53: 154 (1970)).Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-lambda represent expression vectors with the tac, trp, or P_(L)promoters that are currently available (Pharmacia Biotechnology).

A preferred vector is pET21a. This vector is driven by the T7 promoterand is available, for example, from Novagene, Inc. and described inStudier et al., Methods Enzymol., 185: 60-89 (1990). Other preferredvectors are pR1T5 and pR1T2T (Pharmacia Biotechnology). These vectorscontain appropriate promoters followed by the Z domain of protein A,allowing genes inserted into the vectors to be expressed as fusionproteins. Another suitable vector is pB0475, which contains origins ofreplication for phage and E. coli that allow it to be shuttled betweensuch hosts, thereby facilitating both mutagenesis and expression(Cunningham et al., Science, 243: 1330-1336 (1989); U.S. Pat. No.5,580,723).

Other preferred vectors can be constructed using standard techniques bycombining the relevant traits of the vectors described above. Relevanttraits include the promoter, the ribosome-binding site, the decorsin orornatin gene or gene fusion (the Z domain of protein A and decorsin oromatin and its linker), the antibiotic resistance markers, and theappropriate origins of replication.

The host cell may be prokaryotic or eukaryotic. Prokaryotes arepreferred for cloning and expressing DNA sequences to produce the fusionprotein. For example, E. coli K 12 strain 294 (ATCC No. 31446) may beused as well as E. coli B, E. coli X1776 (ATCC No. 31537), and E. colic600 and c600hfl, E. coli W3110 (F-, gamma-, prototrophic/ATCC No.27325), bacilli such as Bacillus subtilis, and other enterobacteriaceaesuch as Salmonella typhimurium or Serratia marcesans, and variousPseudomonas species. The preferred prokaryote is E. coli BL21(Stratagene), which is deficient in the OmpT and Lon proteases, whichmay interfere with isolation of intact recombinant proteins, and usefulwith T7 promoter-driven vectors, such as the pET vectors. Anothersuitable prokaryote is E. coli W3110 (ATCC No. 27325). When expressed byprokaryotes the peptides typically contain an N-terminal methionine or aformyl methionine and are not glycosylated. In the case of fusionproteins, the N-terminal methionine or formyl methionine resides on theamino terminus of the fusion protein or the signal sequence of thefusion protein. These examples are, of course, intended to beillustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forfusion-protein-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louven court et al., J.Bacteriol., 154(2):737-742 (1983)), K fragilis(ATCC 12,424), K.bulgaricus (ATCC No. 16,045), K.Wickeramii (ATCC No. 24,178), K. waltii(ATCC No. 56,500), K. drosophilarum (ATCC No. 36,906; Van den Berg etal., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K.marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida;Trichoderma reesia (EP 244,224); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.nidulans (Ballance etal., Biochem. Biophys. Res. Commun., 112:284-289(1983); Tilbum et al., Gene, 26:205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA, 81: 1470-1474 (1984)) and A. niger (Kelly andHynes, EMBO J., 4:475-479 (1985)). Methylotropic yeasts are suitableherein and include, but are not limited to, yeast capable of growth onmethanol selected from the genera consisting of Hansenula, Candida,Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list ofspecific species that are exemplary of this class of yeasts may be foundin C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Other suitable host cells for the expression of fusion proteins arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC No. CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC No. CCL75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC No. CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

Preferably, the Z-domain portion of the fusion protein can be secretedby the cell (has a signal sequence, for example), making it possible toisolate and purify the fusion protein from the culture medium andeliminating the necessity of destroying the host cells that arises whenthe desired peptide remains inside the cell. Alternatively, the fusionprotein can be expressed intracellularly. It is useful to use fusionproteins that are highly expressed.

The peptide may or may not be properly folded when expressed as a fusionprotein. Also, the specific peptide linker containing the cleavage sitemay or may not be accessible to the protease. These factors determinewhether the fusion protein must be denatured and refolded, and if so,whether these procedures are employed before or after cleavage.

When denaturing and refolding are needed, typically the peptide istreated with a chaotrope, such a guanidine HCl, and is then treated witha redox buffer, containing, for example, reduced and oxidizeddithiothreitol or glutathione at the appropriate ratios, pH, andtemperature, such that the peptide is refolded to its native structure.

b. Svnthetic Preparation

When peptides are not prepared using recombinant DNA technology, theyare preferably prepared using solid-phase synthesis, such as thatgenerally described by Merrifield, J. Am. Chem. Soc., 85: 2149 (1963),although other equivalent chemical syntheses known in the art areemployable. Solid-phase synthesis is initiated from the C-terminus ofthe peptide by coupling a protected α-amino acid to a suitable resin.Such a starting material can be prepared by attaching anα-amino-protected amino acid by an ester linkage to a chloromethylatedresin or a hydroxymethyl resin, or by an amide bond to a BHA resin orMBRA resin. The preparation of the hydroxymethyl resin is described byBodansky et al., Chem. Ind. (London), 38: 1597-1598 (1966).Chloromethylated resins are commercially available from BioRadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., “Solid PhasePeptide Synthesis ” (Freeman & Co., San Francisco 1969), Chapter 1, pp.1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan unsubstituted amide at the C-terminus.

The amino acids are coupled to the peptide chain using techniques wellknown in the art for the formation of peptide bonds. One method involvesconverting the amino acid to a derivative that will render the carboxylgroup more susceptible to reaction with the free N-terminal amino groupof the fusion protein. For example, the amino acid can be converted to amixed anhydride by reaction of a protected amino acid withethylchloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, pivaloyl chloride or like acid chlorides.Alternatively, the amino acid can be converted to an active ester suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole.

Another coupling method involves use of a suitable coupling agent suchas N,N′-dicyclohexylcarbodiimide or N,N′-diisopropylcarbodiimide. Otherappropriate coupling agents, apparent to those skilled in the art, aredisclosed in E. Gross & J. Meienhofer, The Peptides: Analysis,Structure, Biology, Vol. I: Major Methods of Peptide Bond Formation(Academic Press, New York, 1979).

It should be recognized that the α-amino group of each amino acidemployed in the peptide synthesis must be protected during the couplingreaction to prevent side reactions involving their active α-aminofunction. It should also be recognized that certain amino acids containreactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, and hydroxyl) and that such functional groups must also beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at that site during both the initial and subsequentcoupling steps. Suitable protecting groups, known in the art, aredescribed in Gross and Meienhofer, The Peptides: Analysis, Structure,Biology, Vol.3: “Protection of Functional Groups in Peptide Synthesis”(Academic Press, New York, 1981).

In the selection of a particular side-chain protecting group to be usedin synthesizing the peptides, the following general rules are followed.An α-amino protecting group (a) must render the α-amino function inertunder the conditions employed in the coupling reaction, (b) must bereadily removable after the coupling reaction under conditions that willnot remove side-chain protecting groups and will not alter the structureof the fusion protein, and (c) must eliminate the possibility ofracemization upon activation immediately prior to coupling. A side-chainprotecting group (a) must render the side-chain functional group inertunder the conditions employed in the coupling reaction, (b) must bestable under the conditions employed in removing the α-amino protectinggroup, and (c) must be readily removable upon completion of the desiredamino acid peptide under reaction conditions that will not alter thestructure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivitywith the agents employed for their removal. For example, certainprotecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl. adamantyl-oxycarbonyl, andp-methoxybenzyloxycarbonyl, are less labile and require moderatelystrong acids, such as trifluoroacetic, hydrochloric, or borontrifluoride in acetic acid, for their removal. Still other protectinggroups, such as benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require stronger acids,such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetatein trifluoroacetic acid, for their removal. Among the classes of usefulamino-acid protecting groups are included:

(1) for an α-amino group, (a) aromatic urethane-type protecting groups,such as fluorenylmethyloxycarbonyl (FMOC) CBZ, and substituted CBZ, suchas, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,2,6-dichlorobenzyloxycarbonyl, and the like; (b) aliphatic urethane-typeprotecting groups, such as BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like;(c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;and d) allyloxycarbonyl. The preferred α-amino protecting groups are BOCor FMOC.

(2) for the side-chain amino group present in Lys, protection may be byany of the groups mentioned above in (1) such as BOC,p-chlorobenzyloxycarbonyl, etc.

(3) for the guanidino group of Arg, protection may be by nitro, tosyl,CBZ, adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,3,6-trimethyl4-methoxyphenylsulfonyl, or BOC.

(4) for the hydroxyl group of Ser, Thr, or Tyr, protection may be, forexample, by C1-C4 alkyl, such as t-butyl; benzyl (BZL); substituted BZL,such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl,and 2,6-dichlorobenzyl.

(5) for the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using groups such as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

(6) for the imidazole nitrogen of His, the tosyl moiety is suitablyemployed.

(7) for the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl,or 2,6-dichlorobenzyl is suitably employed. The preferred protectinggroup is 2,6-dichlorobenzyl.

(8) for the side-chain amino group of Asn or Gin, xanthyl (Xan) ispreferably employed.

(9) for Met, the amino acid is preferably left unprotected.

(10) for the thio group of Cys, p-methoxybenzyl is typically employed.

An appropriately selected protecting group, in the case of Lys, BOC,protects the C-terminal amino acid, e.g., Lys, at the N-amino position.The BOC-Lys-OH can be first coupled to the benzyhydrylamine orchloromethylated resin according to the procedure set forth in Horiki etal., Chemistry Letters, 165-168 (1978) or using isopropylcarbodiimide atabout 25EC for 2 hours with stirring. Following the coupling of theBOC-protected amino acid to the resin support, the α-amino protectinggroup is removed, as by using trifluoroacetic acid (TFA) in methylenechloride or TFA alone. The deprotection is carried out at a temperaturebetween about OEC and room temperature. Other standard cleavingreagents, such as HCl in dioxane, and conditions for removal of specificα-amino protecting groups are described in the literature.

After removal of the α-amino protecting group, the remaining α-amino andside-chain protected amino acids are coupled stepwise within the desiredorder. As an alternative to adding each amino acid separately in thesynthesis, some may be coupled to one another prior to addition to thesolid-phase synthesizer. The selection of an appropriate couplingreagent is within the skill of the art. Particularly suitable as acoupling reagent is N,N′-dicyclohexyl carbodiimide ordiisopropylcarbodiimide.

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in excess, and the coupling is suitably carried outin a medium of dimethylformamide (DMF) or CH₂Cl₂ or mixtures thereof. Ifincomplete coupling occurs, the coupling procedure is repeated beforeremoval of the N-amino protecting group prior to the coupling of thenext amino acid. The success of the coupling reaction at each stage ofthe synthesis may be monitored. A preferred method of monitoring thesynthesis is by the ninhydrin reaction, as described by Kaiser et al.,Anal. Biochem, 34: 595 (1970). The coupling reactions can be performedautomatically using well-known methods, for example, a BIOSEARCH 9500®peptide synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting groupsis suitably accomplished simultaneously or stepwise. When the resinsupport is a chloromethylated polystyrene resin, the bond anchoring thepeptide to the resin is an ester linkage formed between the freecarboxyl group of the C-terminal residue and one of the manychloromethyl groups present on the resin matrix. It will be appreciatedthat reagents that are known to be capable of breaking an ester linkageand of penetrating the resin matrix can cleave the anchoring bond.

One especially convenient method is by treatment with liquid anhydroushydrogen fluoride. This reagent not only will cleave the peptide fromthe resin but also will remove all protecting groups. Hence, use of thisreagent will directly afford the fully deprotected peptide. When thechloromethylated resin is used, hydrogen fluoride treatment results inthe formation of the free peptide acids. When the benzhydrylamine resinis used, hydrogen fluoride treatment results directly in the freepeptide amines. Reaction with hydrogen fluoride in the presence ofanisole and dimethylsulfide at 0° C. for one hour will simultaneouslyremove the side-chain protecting groups and release the peptide from theresin.

When it is desired to cleave the peptide without removing protectinggroups, the protected peptide-resin can undergo methanolysis to yieldthe protected peptide in which the C-terminal carboxyl group ismethylated. The methyl ester is then hydrolyzed under mild alkalineconditions to give the free C-terminal carboxyl group. The protectinggroups on the peptide chain then are removed by treatment with a strongacid, such as liquid hydrogen fluoride. A particularly useful techniquefor methanolysis is that of Moore et al., Peptides, Proc. Fifth Amer.Pept. Symp., M. Goodman and J. Meienhofer, Eds., (John Wiley, N.Y.,1977), p. 518-521, in which the protected peptide-resin is treated withmethanol and potassium cyanide in the presence of crown ether.

Another method for cleaving the protected peptide from the resin whenthe chloromethylated resin is employed is by ammonolysis or by treatmentwith hydrazine. If desired, the resulting C-terminal amide or hydrazidecan be hydrolyzed to the free C-terminal carboxyl moiety, and theprotecting groups can be removed conventionally.

It will also be recognized that the protecting group present on theN-terminal α-amino group may be removed preferentially either before orafter the protected peptide is cleaved from the support.

Purification of the polypeptides of the invention is typically achievedusing conventional procedures such as preparative HPLC (includingreversed-phase HPLC) or other known chromatographic techniques such asgel permeation, ion exchange, partition chromatography, affinitychromatography (including monoclonal antibody columns), orcountercurrent distribution.

The peptides of this invention may be stabilized by polymerization. Thismay be accomplished by crosslinking monomer chains with polyfunctionalcrosslinking agents, either directly or indirectly, throughmulti-functional polymers. Ordinarily, two substantially identicalpolypeptides are crosslinked at their C- or N-termini using abifunctional crosslinking agent. The agent is used to crosslink theterminal amino and/or carboxyl groups. Generally, both terminal carboxylgroups or both terminal amino groups are crosslinked to one another,although by selection of the appropriate crosslinking agent thealpha-amino group of one polypeptide is crosslinked to the terminalcarboxyl group of the other polypeptide. Preferably, the polypeptidesare substituted at their C-termini with cysteine. Under conditions wellknown in the art a disulfide bond can be formed between the terminalcysteines, thereby crosslinking the polypeptide chains. For example,disulfide bridges are conveniently formed by metal-catalyzed oxidationof the free cysteines or by nucleophilic substitution of a suitablymodified cysteine residue. Selection of the crosslinking agent willdepend upon the identities of the reactive side chains of the aminoacids present in the polypeptides. For example, disulfide crosslinkingwould not be preferred if cysteine was present in the polypeptide atadditional sites other than the C-terminus. Also within the scope hereofare peptides crosslinked with methylene bridges.

Suitable crosslinking sites on the peptides, aside from the N-terminalamino and C-terminal carboxyl groups, include epsilon-amino groups foundon lysine residues, as well as amino, imino, carboxyl, sulfhydryl andhydroxyl groups located on the side chains of internal residues of thepeptides or residues introduced into flanking sequences. Crosslinkingthrough externally added crosslinking agents is suitably achieved, e.g.,using any of a number of reagents familiar to those skilled in the art,for example, via carbodiimide treatment of the polypeptide. Otherexamples of suitable multi-functional (ordinarily bifunctional)crosslinking agents are found in the literature.

The peptides of this invention also may be conformationally stabilizedby cyclization. The peptides ordinarily are cyclized by covalentlybonding the NB and C-terminal domains of one peptide to thecorresponding domain of another peptide of this invention so as to formcyclo-oligomers containing two or more iterated peptide sequences, eachinternal peptide having substantially the same sequence. Further,cyclized peptides (whether cyclo-oligomers or cyclo-monomers) arecrosslinked to form 1-3 cyclic structures having from 2 to 6 peptidescomprised therein. The peptides preferably are not covalently bondedthrough α-amino and main-chain carboxyl groups (head to tail), butrather are crosslinked through the side chains of residues located inthe NB and C-terminal domains. The linking sites thus generally will bebetween the side chains of the residues.

Many suitable methods per se are known for preparing mono- orpoly-cyclized peptides as contemplated herein. Lys/Asp cyclization hasbeen accomplished using sodium-tert-butyloxycarbonyl (Na-Boc)-aminoacids on solid-phase support with Fmoc/9-fluorenylmethyl (OFm)side-chain protection for Lys/Asp; the process is completed bypiperidine treatment followed by cyclization.

Glu and Lys side chains also have been crosslinked in preparing cyclicor bicyclic peptides: the peptide is synthesized by solid-phasechemistry on a p-methylbenzhydrylamine resin. The peptide is cleavedfrom the resin and deprotected. The cyclic peptide is formed usingdiphenylphosphorylazide in diluted methylformamide. For an alternativeprocedure, see Schiller et al., Peptide Protein Res., 25: 171-177(1985). See also U.S. Pat. No. 4,547,489

Disulfide crosslinked or cyclized peptides are generated by conventionalmethods. The method of Pelton et al. (J. Med. Chem., 29: 2370-2375(1986)) is suitable, except that a greater proportion of cyclo-oligomersare produced by conducting the reaction in more concentrated solutionsthan the dilute reaction mixture described by Pelton et al., for theproduction of cyclo-monomers. The same chemistry is useful for synthesisof dimers or cyclo-oligomers or cyclo-monomers. Also useful arethiomethylene bridges. LebI and Hruby, Tetrahedron Letters, 25:2067-2068 (1984). See also Cody et al., J. Med. Chem., 28: 583 (1985).

The desired cyclic or polymeric peptides are purified by gel filtrationfollowed by reversed-phase high-pressure liquid chromatography or otherconventional procedures. The peptides are sterile filtered andformulated into conventional pharmacologically acceptable vehicles.

The starting materials required for the processes described herein areknown in the literature or can be prepared using known methods and knownstarting materials.

If in the peptides being created carbon atoms bonded to fournonidentical substituents are asymmetric, then the peptides may exist asdiastereoisomers, enantiomers or mixtures thereof. The synthesesdescribed above may employ racemates, enantiomers or diastereomers asstarting materials or intermediates. Diastereomeric products resultingfrom such syntheses may be separated by chromatographic orcrystallization methods. Likewise, enantiomeric product mixtures may beseparated using the same techniques or by other methods known in theart. Each of the asymmetric carbon atoms, when present, may be in one oftwo configurations R) or S) and both are within the scope of the presentinvention.

In another embodiment, the fragment of the fusion protein herein may beaffinity matured so that it has better affinity toward the IGF-I and/orIGF-II than the parent fragment. Such affinity maturation can be done,for example, through phage display, rational mutagenesis, randommutagenesis, or DNA shuffling and phage display, or by any such othermeans known in the art for effecting this change. See the referencesnoted above in the definition section.

2. Uses

There are many advantages to using the fusion proteins for theapplications disclosed herein. For example, mammalian systems can beexpensive for the industrial production of wild-type IGFBP-3. On theother hand, peptide fusions are expected to be cheaper and easier toproduce than wild-type IGFBP-3 using either synthetic chemical methodsor highly efficient biological production systems well known to thoseskilled in the art.

The peptides herein may be useful in diagnostic assays, e.g., fordetecting expression of IGF-1 in specific cells, tissues, or serum.

For diagnostic applications, the peptide typically will be labeled witha detectable moiety. Numerous labels are available that can be generallygrouped into the following categories:

(a) Radioisotopes, such as 35S, I4C, I211, 3H, and 131I, are available.The peptide can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., ed. (Wiley-lnterscience: New York, 1991), for example, andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare-earth chelates (europium chelates)or fluorescein and its derivatives, rhodainine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the peptide using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate that can bemeasured using various techniques. For example, the enzyme may catalyzea color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light that can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,“Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay,” in Methods in Enzym. (ed J. Langone & H. VanVunakis), 73:147-166 (Academic Press, New York, 1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) beta-D-galactosidase (beta-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-beta-D-galactosidase) or fluorogenic substrate(4-methylumbellifuryl-beta-D-galactosidase).

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the peptide. Theskilled artisan will be aware of various techniques for achieving this.For example, the peptide can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the peptide in this indirect manner.Alternatively, to achieve indirect conjugation of the label with thepeptide, the peptide is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten peptide (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the peptide can be achieved.

In another embodiment of the invention, the peptide need not be labeled,and the presence thereof can be detected using a labeled antibody thatbinds to the peptide.

The peptide of the present invention may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The peptide may also be used for in vivo diagnostic assays. Generally,the peptide is labeled with a radionuclide (such as 111In, 99Tc, 14C,131I, 125I, 3H, 32P or 35S) so that the antigen or cell localized usingimmunoscintiography.

Other uses include mapping binding epitopes of bp15 and IGFBP-3 andother IGF peptide agonists. In addition, the IGFBP-3 fragments that bindto IGF-I or IGF-II or fusion proteins comprising them can be used incell-based assays. Certain such assays comprise contacting the cell withthe fusion protein rather than with native-sequence human IGFBP-3 anddetermining if a biological activity attributable to native-sequencehuman IGFBP-3, native-sequence human IGF-I or native-sequence IGF-II, oran agonist of said IGF-I or said IGF-II is observed. In one embodiment,the biological activity is apoptosis of native-sequence human IGFBP-3that is independent of IGF-I. In another example, the assay is anIGF-dependent KIRA phosphorylation assay. This assay is an IGF-I kinasereceptor activation assay, which is a direct activity assay for thehuman Type 1 receptor. When a receptor in the tyrosine kinase family,such as the Type 1 IGF receptor, is activated, it is phosphorylated ontyrosine residues. In this assay cells containing the Type 1 IGFreceptor are activated in vitro, then disrupted, and antibodies againstthe receptor are used to precipitate the IGF receptor. Next, ananti-phosphotyrosine antibody is used to assay the amount of Type 1 IGFreceptor that is phosphorylated. If a fixed number of cells are used,then the amount of receptor that is phosphorylated is a direct measureof the activity of a molecule on the Type 1 IGF receptor. In this KIRAassay, cells such as a breast cancer cell line are treated with IGF-I orIGF-II plus the fusion protein and a biological activity of the fusionprotein is determined by the amount of receptor that is phosphorylated.KIRA assays are described further in U.S. Pat. No. 6,251,865 using MCF-7breast cancer cells as one embodiment, and in Chen et al., Am. J.Physiol. Endocrinol. Metab., 284: E1149-E1155 (2003).

In yet another embodiment, the biological activity is inhibition ofbinding of radiolabeled IGF-I or IGF-II to the cells.

A further example is a method comprising pre-treating breast cancercells with an IGFBP-3 fragment or fusion protein thereof that binds toIGF-I or IGF-II for at least about 24 hours prior to treating the cellswith an apoptotic factor, such as, for example, a chemotherapeuticagent, e.g., doxorubicin or paclitaxel, and native-sequence humanIGFBP-3 or said IGFBP-3 fragment or fusion protein, and determining ifthe pre-treatment or treatment enhances the apoptosis induced by thetreatment with apoptotic factor, or if the amounts of pre-treatment ortreatment are effective for that purpose.

Kits are also contemplated for this invention. The kit generallycomprises a container containing a composition comprising the fusionprotein and instructions for its use, e.g., in an assay. A typical kitwould comprise a container, preferably a vial, for the fusion proteinformulation comprising the fusion protein in a buffer and instructions,such as a product insert or label, directing the user to utilize theformulation, e.g., for mapping epitopes or cell-based assays. The kitoptionally includes a container, preferably a vial, for an agent to beused with the fusion protein.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLE 1 Production of IGFBP-3 and MiniBP-3 Fusion Protein

Introduction:

Native-sequence human IGFBP-3 and miniBP-3 fusion protein were preparedand assessed for direct binding to native-sequence human IGF-I andIGF-II in BIAcore™ assays.

Based upon the results of experiments using the miniBP-3 fusion proteindescribed below, it is predicted that molecules of the type claimedherein should decrease active IGF levels.

Materials and Methods:

Expression and Purification of Wild-type IGFBP-3 and MiniBP-3 Fusion andCleaved MiniBP-3

Wild-type IGFBP-3 and miniBP-3 were produced in E. coli using the vectorpET21a (Novagene) as follows:

All routine chemical reagents were purchased from Sigma Chemical Co. (StLouis, Mo.) or Fisher Scientific (Fair Lawn, N.J.). Restriction enzymesand the T4 DNA ligase were obtained from New England Biolabs (Beverley,Mass.). Oligonucleotide synthesis reagents, DNA sequencing kits, and PCRkits were obtained from PE Biosystems (Foster City, Calif.). dNTPs, IPTGand ATP were purchased from Boehringer-Mannheim (Indianapolis, Ind.).DNA polymerase and E. coli strain BL21 were purchased from Stratagene(La Jolla, Calif.).

Plasmid pET21a was purchased from Novagen Inc. (Madison, Wis.). Theaffinity column was from Pharmacia (Piscataway, N.J.). LB medium wasprepared according to the standard formula (Sambrook et al., supra).HEPES and CHAPS buffers and DTT are obtainable from the sourcesindicated below. Periplasmic extraction buffer consisted of 10 mMTRIS-HCI, pH 8.5 and 1 mM EDTA. TE buffer consisted of 10 mM TRIS-HCl,pH 8.0 and 1 mM EDTA.

Oligodeoxyribonucleotides were synthesized using a 394 automated DNAsynthesizer from PE Biosystems. PCR and sequencing primers were purifiedby ethanol precipitation and dissolved in TE buffer. The Z domain wasamplified by PCR of vector pA-100-Z (derived from the construct asdescribed in Dennis et al., Biochemistry, 40: 9513 (2001)) using theprimers: 5′-ACT AAA TAT GCT AGC GCC GTA GAC AAC AAA TTC AAC AAA G-3′(SEQ ID NO:13) and 5′-ATA TTT AGT GAA TTC CTT AGG CGC CTG AGC ATC ATTTAG-3′ (SEQ ID NO: 14). The amplified Z domain was digested with NheIand EcoRI and ligated into the cloning vector pET21a (Novagen) treatedwith the same pair of restriction enzymes to generate pET21a-Z domainfusion. The insertion of the Z domain was confirmed by sequencing.

The coding region of miniBP-3 fusion was synthesized by PCR using thegene assembly method of Stemmer et al., Gene, 164: 49-53 (1995). Theoligonucleotides used for gene assembly had the sequences: 5′-ACT AAATAG AAT TCG GCG GTG ATG ATG ACG ACA AAG CCC TGA GCG AAG GTC AGC CGT GCGGTA TTT AT-3′ (SEQ ID NO: 15), 5′-GCT CGG CTG GCA ACG CAG ACC GCT ACCGCA ACG TTC GGT ATA AAT ACC GCA CGG-3′ (SEQ ID NO:16),5 ′-CGT TGC CAGCCG AGC CCG GAT GAA GCC CGT CCG CTG CAG GCC CTG CTG GAT GGT CGT GGT CTGTGC-3′ (SEQ ID NO:17), and 5′-TAT TTA GTA AGC TTC TAA TAG GCA CGC AGACGG CTA ACG GCG CTG GCG TTA ACG CAC AGA CCA CGA CC-3′ (SEQ ID NO:18).The assembled gene was amplified using the following oligonucleotides:5′-ACT AAA TAG AAT TCG GCG GTG ATG-3′ (SEQ ID NO:19) and 5′-TAT TTA GTAAGC TTC TAA TAG GCA CG-3′ (SEQ ID NO:20).

The gene encoding full-length fusion miniBP-3 protein was amplified withthe primers using a PCR System 9700™ thermocycler (PE Biosystems) asdescribed in Cao et al., Gene, 197: 205-214 (1997). The amplifiedproduct was digested with Eco RI and Hind III and ligated into thepET21a-Z-domain-containing vector treated with the same pair ofrestriction enzymes to generate pET21a-miniBP-3 fusion. The insertion ofthe miniBP-3 fragment was confirmed by sequencing. The enterokinasecleavage site EFGGDDDDK (SEQ ID NO:4) was later replaced with thecaspase-3 cleavage site EFGGDLVD (SEQ ID NO:7) using the QUIKCHANGE™Site-Directed Mutagenesis Kit. These two mutagenic primers had thesequences: 5′-GGA ATT CGG CGG TGA TCT GGT GGA TGC CCT GAG CGA AGG-3′(SEQ ID NO:21) and 5′-CCT TCG CTC AGG GCA TCC ACC AGA TCA CCG CCG AATTCC-3′ (SEQ ID NO: 22). The mutation was confirmed by sequencing.

This vector was transformed into E. coli strain BL21 (Stratagene).Inserts in pET expression vectors were sequenced in both orientations toensure that the plasmid constructs were free of PCR or ligation errors.

The cells were propagated overnight at 37° C. in 2YT medium (Sambrook etal, supra) containing 50 μg/ml of ampicillin. Overnight cultures werediluted 100-fold into the same medium, grown until the optical densityof the culture reached 0.5 at 600 nm, then induced by the addition ofisopropyl-beta-D-thiogalactopyranoside (IPTG) to a final concentrationof 50 μM and grown for an additional 4 hours under the same conditions.Cells were collected by centrifugation, frozen/thawed at −20/4° C. inperiplasmic extraction buffer, and clarified by centrifugation asdescribed by Cao et al., supra.

The periplasmic extracts from the centrifugation were subjected tochromatography using an IgG SEPHAROSE™ ion-exchange column (AmershamBiosciences) as described in Nilssen et al., supra. Briefly, the columnwas washed with 5 volumes 50 mM TRIS buffer, pH 7.6, 150 mM NaCl and0.05% TWEEN-20™ buffer (TST), then alternately with 0.5 M acetic acid(HAc), pH 3.4 and TST for two cycles. The column was equilibrated withTST and the fusion protein was captured on the column, as described inNilsson et al., supra. The column was washed with 10 bed volumes TST and2 bed volumes of 5 mM ammonium acetate, pH 5.0 before elution.

After the fusion protein was eluted with 4 bed volumes of 0.5M HAc itwas incubated overnight at 4° C. with 125 nM caspase-3 (kindly providedby Dr. Guy Salvesen, The Burnham Institute, La Jolla, Calif.) in 100 mMHepes buffer (ProSciTech), 0.1% CHAPS lysis buffer (ChemiconInternational), 0.5 mM dithiothreitol (DTT) (JT Baker), pH 7.5. CleavedminiBP-3 was recovered from the supernatant of the reaction aftercentrifugation. The purity of the fusion protein was verified bySDS-PAGE analysis followed by visualizing the overloaded gel withroutine Coomassie brilliant blue R staining. See FIG. 2, which shows theSDS-PAGE analysis for the various stages.

BL21 cells expressing wild-type IGFBP-3 were cultured and expression wasinduced as with miniBP-3. The wild-type IGFBP-3 was extracted frominclusion bodies and refolded in vitro using standard conditions.Purification was achieved using ion-exchange chromatography on both Qand S SEPHAROSE™ (Amersham Biosciences) columns andhydrophobic-interaction chromatography on a phenyl SUPEROSE™ (AmershamBiosciences) column using standard techniques.

Biosensor Kinetic Measurements

The binding affinities of wild-type IGFBP-3 and miniBP-3 fusion proteinfor IGF-I and IGF-II were determined using a BIAcore™-2000 real timekinetic interaction analysis system (BIAcore, Inc., Piscataway, N.J.) tomeasure association (K_(a)) and dissociation (K_(d)) rates. Two types ofchips were prepared for this purpose.

CM5 Chip Preparation:

Carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) wereactivated with EDC (N-ethyl-N′-3-dimethylaminopropyl) -carbodiimidehydrochloride) and NHS (N-hydroxysuccinimide) according to thesupplier's instructions. For immobilization, wild-type IGFBP-3 andmutants in 10 mM sodium acetate, pH 4, were injected onto the biosensorchip at a concentration of 0.2 mg/ml to yield approximately 450-700 RU's(resonance-response units) of covalently coupled protein. Unreactedgroups were blocked with an injection of 1 M ethanolamine.

Kinetic measurements were carried out by injecting two-fold serialdilutions of IGF-I or IGF-II in PBST running buffer (PBS, 0.05%TWEEN-20™ buffer, 0.01% sodium azide) at 25° C. using a flow rate of 20or 50:1/min IGF concentrations ranged between 0.4 nM and 50 nM forIGFBP-3 and 100 nM to 25:M for miniBP-3 fusion. For both proteins,association and dissociation rates were calculated using a 1:1 Langrnuirassociation model in the BIAcore™ evaluation software. For wild-typeIGFBP-3, the equilibrium dissociation constant was calculated ask_(d)/k_(a). For miniBP-3 fusion, the equilibrium dissociation constantwas calculated by plotting equilibrium binding data in GraphPad Prism™software (GraphPad Software Inc., San Diego, Calif.) and fitting to aone-site binding model.

SA Chip Preparation:

Streptavidin-coated chips were conditioned according to themanufacturer's instructions (BIAcore, Inc.) prior to injection of 0.02mg/ml of biotinylated IGF-I or IGF-II in 10 mM sodium acetate pH 4.Biotinylated IGF-I and IGF-II were prepared using EZ-Link biotinylationreagents according to the manufacturer's instructions (Pierce). Between100 and 1000 RUs were immobilized on the chip.

Kinetic measurements were carried out by injecting two-fold serialdilutions of miniBP-3 fusion protein in PBST running buffer at 50:1/min.Concentrations were between 250 nM and 32:M.

Competition binding experiments were carried out as follows. 15 nM to100:M bp15 peptide and 20 nM IGFBP-3 were incubated for 1 hour at roomtemperature before injection at 20:1/min in PBST over immobilizedbiotinylated IGF-I and -II. Equilibrium binding data was plotted inGraphPad Prism™ software and fit to a one-site competition-bindingmodel.

Results:

The wild-type IGFBP-3 and miniBP-3 fusion protein were submitted forkinetic analysis using a BIAcore™ instrument and tested for bindingaffinity to IGF-I and IGF-II. The results are shown in Table I forwild-type IGFBP-3 and Table II for miniBP-3 fusion protein.

TABLE I Kinetic Parameters for the Interaction of Purified Wild-typeIGFBP-3 with IGF-I and -II Determined by BIAcore ™ Analysis k_(a) (×10⁴M⁻¹ s⁻¹) k_(d) (×10⁻⁴ s⁻¹) K_(D) (×10⁻⁹ M) IGF-I 715 ± 3.66 7.58 ± 0.070.11 ± 0.001 IGF-II 348 ± 1.02 4.07 ± 0.05 0.12 ± 0.001

TABLE II Kinetic Parameters for the Interaction of Purified MiniBP-3Fusion Protein with IGF-I and -II Determined by BIAcore ™ Analysis k_(a)(×10⁴ M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (×10⁻⁶ M) IGF-I 9.13 ± 0.87 0.59 ± 0.044.64 ± 0.17 IGF-II 7.59 ± 1.35 0.08 ± <0.01 4.66 ± 0.06

The results in Table I (see also FIGS. 3 and 4), showing that IGFBP-3has high affinity for IGF-I and IGF-II, compare favorably with othermeasurements in the literature. The results in Table II (see also FIGS.5 and 6) as compared to Table I show that the miniBP-3 fusion has loweraffinity for IGF-I and IGF-II compared to wild-type IGFBP-3. Withoutbeing limited to any one theory, it is believed that this is primarilydue to increases in off-rate. Studies with other fragments of IGFBP-3display similar findings, as shown in Table III.

TABLE III Kinetic Parameters for the Interaction of Purified N-TerminalFragments of IGFBP-3 with IGF-I and -II Determined by BIAcore ™ AnalysisFragment k_(a) (×10⁴ M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(A) (×10⁵ M⁻¹) ReferenceN-88 14.7 ± 1.6 1.26 ± 0.28 × 10⁻²  103 ± 8 Galanis et al. (2001), supraIGFBP-3¹⁻⁹⁷  3.2 ± 1.1 5.55 ± 1.40 × 10⁻³ 61.4 ± 19.8 Vorwerk et al.(2002), supra

EXAMPLE 2 Binding of Peptide bp15 to MiniBP-3 Fusion

Peptide bp15 (SEEVCWPVAEWYLCN) (SEQ ID NO:11) was identified by phagedisplay (Lowman et al., Biochemistry, 1998, supra). It competes withIGF-I and -II for binding to IGFBP-3 as determined by BIAcore™ analysisas described in Example I. Therefore, bp15 was tested to see if it wouldbind to the miniBP-3 fusion. Compare FIGS. 7 and 8 for competitivebinding data for bp15 to IGFBP-3 and to the miniBP-3 fusion,respectively.

It can be seen that peptide bp15 does bind to the miniBP-3 fusion, butwith reduced affinity compared to wild-type IGFBP-3. Exact affinity wasunable to be determined due to lack of saturation.

Discussion:

The miniBP-3 fusion has affinity for IGF-I and IGF-II. This affinity isreduced compared to that of other N-terminal fragments of IGFBP-3.Nevertheless, the miniBP-3 fusion contains at least part of the bp15peptide-binding site. N-terminal fragments of IGFBP3 have also beenassociated with IGF-independent effects of IGFBP-3. See, for example,Angelloz-Nicoud et al., Growth Hormone & IGF Research, 8: 71-75 (1990);Lalou et al., Endocrinology, 137 (8): 3206-3212 (1996); Yamanaka et al.,Endocrinolgy, 140 (3): 1319-1328 (1999 Endocrinology, 140(9): 4040-4045(1999); Salahifar et al., Growth Hormone & IGF Research, 10: 367-377(2000); and Bernard et al., Biochem. Biophys. Res. Comm., 293: 55-60(2002).

Cell-based assays using a fusion protein containing miniBP-3 or anIGFBP-3 fragment can be developed to detect these IGF-independentevents. Thus, in experiments performed by Gill et al., J. Biol. Chem.,272: 25602-25607 (1997) and Fowler et al., Int. J. Cancer, 88: 448-453(2000), breast cancer cells are treated with IGFBP-3 for 24 hours priorto the addition of paclitaxel (and IGFBP-3). Although treatment of thecells with IGFBP-3 alone is not sufficient to induce apoptosis,pre-treatment with IGFBP-3 enhances the apoptosis induced by paclitaxeltreatment. The miniBP-3 fusion protein, or fragments of IGFBP-3 thatbind to IGF-I or IGF-II and fusion proteins thereof, are expected to beuseful in this assay instead of wild-type IGFBP-3 if they contain theactivity responsible for potentiation of cell death. An advantage ofusing the fusion protein or fragments is that they are easier to make inlarge quantities than intact IGFBP-3, as noted above.

Additionally to these apoptosis assays, cell-based assays can be usedfor IGF-dependent KIRA assays and radiolabeled IGF binding inhibition,because wild-type IGFBP-3 does not inhibit binding on some cells,perhaps, without being limited to any one theory, due to binding to itsown receptor. In an exemplary cell-based IGF-1 KIRA assay, a KIRA formeasuring the activation of the human type 1 IGF-1 receptor is performedusing human MCF-7 cells. Cells are grown overnight in 96-well plateswith medium (50:50 F12/DMEM, Gibco). Supernatants are decanted, andstimulation media (50:50 F12/DMEM with 25 mM HEPES and 2.0% BSA)containing either controls (2 nM IGF-1 preincubated with wild-typeIGFBP-1 or IGFBP-3) or experimental samples (fusion protein containingminiBP-3 preincubated for30 min. with 2 nM IGF-1 ) are added. After15-minute stimulation the cells are lysed, and added to a polyclonalanti-IGF-1R (3B7; Santa Cruz Biotech) coated overnight on immunosorbantplates. Detection ELISA is performed, and the KIRA results would beexpected to show about the same inhibition for the fusion proteincontaining miniBP-3 as, or possibly more inhibition of IGF-I receptorbinding than, that seen for wild-type IGFBP-3, and the inhibition islikely to be improved if the miniBP-3 fragment is affinity matured byphage display or other means as would be known to those skilled in theart.

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the objectives of the presentinvention.

1. A method for monitoring enhancement of apoptosis comprisingpre-treating breast cancer cells with an IGFBP-3 fragment or fusionprotein thereof that binds IGF-I or IGF-II for at least about 24 hoursprior to treating the cells with an apoptotic factor and native-sequencehuman IGFBP-3 (SEQ ID NO: 1) or said IGFBP-3 fragment or fusion protein,and determining if the pre-treatment or treatment enhances the apoptosisinduced by the treatment with the apoptotic factor.
 2. The method ofclaim 1 wherein the apoptotic factor is doxorubicin or paclitaxel. 3.The method of claim 1 wherein the fusion protein comprises a peptide ofSEQ ID NO: 10 linked to said IGFBP-3 fragment.
 4. The method of claim 3wherein the IGFBP-3 fragment is a fragment of residues 47 to 99 ofnative-sequence human IGFBP-3 (SEQ ID NO:1).